Fraction of melissa leaf extract having angiogenesis and mmp inhibitory activities, and composition comprising the same

ABSTRACT

The present application describes an ethyl acetate fraction of Melissa leaf having excellent angiogenesis and MMP inhibitory activities, and a composition comprising the same.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. application Ser. No.12/609,516, filed Oct. 30, 2009, which is a continuation-in-part ofPCT/KR2008/004938, filed Aug. 25, 2008, the contents of each areincorporated by reference herein in their entirety.

BACKGROUND OF THE INVENTION 1. Field of the Invention

The present invention relates to an ethyl acetate fraction of Melissaleaf having excellent angiogenesis and MMP inhibitory activities, and acomposition comprising the same. In particular, the ethyl acetatefraction of Melissa leaf is characterized in that Melissa leaf isextracted with 50˜100% C₁˜C₆ alcohol, and concentrated, and then theconcentrated alcohol extract is suspended in water, and fractionatedwith ethyl acetate, and dried to obtain the ethyl acetate fraction ofMelissa leaf.

2. General Background and State of the Art

Melissa (Melissa officinalis), a perennial herb in a Labiatae family, isalso called lemon balm, balm, or dropsy plant as common and folk names.

Key constituents of the Melissa officinalis are flavonoids, terpeneacids, volatile oils, glycosides of the alcoholic and phenolic compoundsand caffeic acid derivatives. Especially, cynaroside, cosmosin,rhamnocitrin and isoquercitrin are abundant components as flavonoids,and ursolic acid is a component as terpene acid. Rosmarinic acid ascaffeic acid derivatives is the most abundant component (about 4.7%),and geranial, neral, citronellal and eugenol are well known componentsas volatile oils contained in Melissa leaf extract.

Rosmarinic acid, an abundant non-volatile component of Melissa leafextract, has strong anti-inflammatory and antipyretic effect, andessential oils have been used for depression, neurogenic headache,reducing memory, neuralgia, fever and also well known to have sedative,antibacterial, antiviral, antioxidant and antihormonal effects.Recently, Melissa leaf extract has included in blood circulationactivator, which helps dilatation of peripheral blood vessels.

Angiogenesis is the process of generating new capillary blood vessels.Neovascularization is tightly regulated, and occurs during embryonicdevelopment, tissue remodeling, wound healing and periodic cycles ofcorpus luteum development (Folkman and Cotran, Relation of vascularproliferation to tumor growth, Int Rev Exp Pathol 16 207-248, 1976).

The endothelial cells grow very slowly as compared with other types ofcells in the body. However, the proliferation of endothelial cells isinduced by pro-angiogenic cytokines, activated hydrolytic enzymes whichrelease the angiogenic mediators from extracellular matrix or thestimulation of angiogenic factors.

Generally, the process of angiogenesis is the degradation of basementmembrane of blood vessels, the migration of endothelial cells, and thelumen formation via proliferation and differentiation of endothelialcells. One of the major events in the process of angiogenesis is abreakdown of the extracellular matrix before the formation of thecapillary blood vessels. The most important enzyme of matrix degradationis matrix metalloproteinase (MMP).

When the failure in the regulation of angiogenesis occurs or MMPs areover activated, pathological angiogenesis takes place, which is relatedto many diseases (Polverini P J, Critical Reviews in Oral Biology, 6(3),230-247(1995); Amp Das, et al., Progress in Retinal and Eye Research, 22(2003) 721-748; Nick Di Girolamo, et al., IOVS, August 2001, Vol. 42,No. 9, 1963-1968; Patricia Lee, et al., Survey of ophthalmology, Vol 43,No. 3, November-December 1998, 245-269; D. B. Holland, et al., BritishJournal of Dermatology 2004, 150, 72-81; Anthony H Vagnucci Jr, et al.,The Lancet, Vol 361, Feb. 15, 2003, 605-608; Berislav V. Zlokovic,Trends in Neuroscience, Vol. 28, No. 4, April 2005, 202-208; Jaap G.Neels, et al., The FASEB Journal express article 10. 1096403-1101fje.Published online Apr. 14, 2004; D. L. Crandall, et al.,Microcirculation, 4, 1997, 211-232; G. Voros, et al., Endocrinology,146, 2005, 4545-4554; M. A. Rupnick, et al., PNAS, 99, 2002,10730-10735; E. Brakenhielm, et al., Circ. Res., 94, 2004, 1579-1588; H.R. Lijnen, et al., Arterioscler Thromb Vasc Biol., 22. 2002, 374-379; D.Demeulemeester, et al., Biochem. Biophys. Res. Commun., 329, 2005,105-110, etc.) such as,

Cancer growth and metastasis;

Angioma, angiofibroma, vascular deformity, and cardiovascular diseasessuch as atherosclerosis, angiostenosis, edemic sclerosis and stenosis;

Opthalmological diseases such as diabetic retinopathy, maculardegeneration (including age-related macular degeneration), pterygium,retinal degeneration, angiogenesis in corneal implantation, angiogenicglaucoma, angiogenic corneal diseases such as corneal synechia and irissynechia, retrolental fibroplasias, granular conjunctivitis, cornealulcer, proliferate vitreous body retinopathy, immature retinopathy, eyeinflammation in eyes, conical cornea, Sjogren's syndrome, myopia, eyestumors and rejection in cornea implantation;

Obesity;

Chronic inflammatory diseases such as arthritis, rheumatoid arthritis,osteoarthritis, septic arthritis, MMP-mediated osteopathy anddegenerative cartilage loss following traumatic joint injury;

Inflammation diseases such as inflammation of the central nervous systemand inflammatory bowel disease;

Dermatological disease such as psoriasis, telangiectasis, pyogenicgranuloma, seborrheic dermatitis and acne;

Alzheimer's disease;

Corneal synechia, proteinuria, abdominal aortic aneurysm, demyelinatednerve tissue, liver fibrosis, nephroglomerular disease, prematurerupture of the fetal membrane, periodontal diseases such as gingivitisand periodontitis.

In particular, angiogenesis plays very important role in growth andmetastasis of cancer cells. New blood vessels supply not only nutrientsand oxygen to fast-growing cancer cells, but also provide access to thecirculation for the evolution of metastasis [Folkman and Tyler, CancerInvasion and metastasis, Biologic mechanisms and Therapy (S. B. Day ed.)Raven press, New York, 94-103(1977); Polverini P J, Critical Reviews inOral Biology, 6(3), 230-247(1995)].

Cancer patients die because of metastasis, and chemotherapy andimmunotherapy currently used in the treatment of cancer can notcontribute to the survival of cancer patients due to the lack ofanti-metastasis effects.

Arthritis, a well-known inflammatory disease, is initiated as anautoimmune disease. However, the growth of vascular endothelial cell inthe synovial cavity is activated by the inflammatory cytokines, whichfinally destroyed cartilage in the articulation. In other words, theproliferation of synovial cells and endothelial cells in synovial cavitywith the help of cytokines inducing inflammation induces angiogenesisand pannus formation, which are the major role in destroying cartilage(Kocb A E, Polverini P J and Lcibovich S J, Arth Rheum 29, 471-479,1986; Stupack D G, Storgard C M and Cheresh D A, Braz J Med Biol Rcs 32,578-581, 1999 Koch A E, Arthritis Rheum 41, 951-962, 1998). Meanwhile,it has also been shown that stromelysin in arthritis and traumatic jointinjury is recognized to play an important role in the activation ofprocollagenase to active collagenase [Murphy, G. et al., Biochem. J.248, 265˜268(1987)]. Therefore, the downregulation of MMP activity canprevent the progress of arthritis.

Many people are losing their eyesight all over the world because ofvarious ocular diseases. Many patients become blind due to theinfiltration of the capillary blood cells into the vitreous humor(Jeffrey M I and Takayuki A, J Clin Invest 103, 1231-1236, 1999).Age-related macular degeneration, diabetic retinopathy, retinopathy ofprematurity, angiogenic glaucoma, angiogenic corneal diseases aretypical angiogenic ocular diseases [Adamis A P, Aiello L P and D'Amato RA, Angiogenesis 3, 9-14(1999)]. Diabetic retinopathy, a complication ofdiabetes, is caused by the rupture of capillaries and by the covering ofhemorrhage on the surface of retina.

Collagenase, gelatinase and stromelysin have been implicated in thedestruction of the extracellular matrix of the cornea. This is thoughtto be an important mechanism of morbidity and visual loss in a number ofulcerative ocular diseases, particularly those following infection orchemical damage (Burns, F. R. et al., Invest Opthalmol and Visual Sci,32, 1569-1575, 1989). The MMPs present in the eye during ulceration arederived either endogenously from infiltrating leucocytes or fibroblasts,or exogenously from microbes.

Psoriasis is caused by extremely active proliferation of keratinocytes.Fast-growing cells require sufficient blood supply, and angiogenesis isabnormally induced in psoriasis (Folkman J., J Invest Dermatol 59,40-48, 1972).

Collagenases, secreted by inflammatory stimulation and microbes,decompose the collagen in gingival connective tissue and finally causeperiodontitis. Collagenase and stromelysin activities have beenidentified in fibroblasts isolated from inflamed gingiva and the levelsof enzyme have been correlated with the severity of the gingivitisobserved (Beeley, N. R. A. et al., supra Overall, C. M. et al., JPeriodontal Res, 22, 81-88, 1987).

MMPs are correlated with the pathogenesis of CNS(Central Nerve System),which destroy myelin or blood-brain barrier. MMP is also reported to berelated to the accumulation of amyloid beta protein in Alzheimer'sdisease [Yong, V W. et al., Trends Neurosci 21(2), 75-80(1998)].

Excessive levels of gelatinase-B in cerebrospinal fluid has been linkedwith incidence of multiple sclerosis and other neurological disorders[Beeley, N. R. A. et al., supra.; Miyazaki, K. et al., Nature 362,839˜841(1993)], and contribute to degradation and the accumulation ofamyloid beta protein [Backstrom J R, et al., J neurosci 16(24), 7910-9(1996)].

Recent reports have also shown that MMP-1 activity is highly induced inthe brain of Alzheimer's disease, and MMP-3 which activates theproenzyme of MMP is also involved in the pathophysiology of the disease(Leake A, Morris C M, & Whateley, J Neurosci Lett 291, 201-3, 2000;Yoshiyama Y, Asahina M, & Hattori T, Acta Neuropathol (berl), 99, 91-5,2000).

The degradation of basement membrane by MMP is very important procedurefor cancer invasion, metastasis and also angiogenesis. So, theoverexpression of MMP can stimulate angiogenesis, cancer invasion andmetastasis.

In the case of adenocarcinoma, invasive proximal gastric cancer cellsexpress the 72-kD form of type IV collagenase [Schwartz, G. K. et al.,Cancer 73, 22˜27(1994)]. Rat embryo cells transformed by the Ha-ras andv-myc oncogenes or by Ha-ras alone are metastatic in nude mice andrelease the 92 kDa gelatinase/collagenase (MMP-9) [Bernhard, E. J. etal., Proc. Natl. Acad. Sci 91, 4293˜4597(1994)].

Therefore, angiogensis inhibitors or MMP inhibitors can be developed forthe therapeutics of these diseases.

In relation to this, the inventors disclosed at KR registration No.10-550,298 that Melissa leaf extract had anti-angiogenic effect throughin vitro assay such as HUVEC (Human Umbilical Vein Endothelial cell)tube formation, and in vivo assay such as mouse Matrigel model and CAMassay.

Furthermore, the inventors disclosed at KR registration No. 10-473,688that Melissa leaf extract had inhibition effect on MMP (matrixmetalloproteinase) which is important to degrade the basement membranein the process of angiogenesis.

The inventors bring to completion this invention through research toobtain more active Melissa leaf fraction having excellent angiogenesisand MMP inhibitory activities.

SUMMARY OF THE INVENTION

It is an objection of the present invention to provide an ethyl acetatefraction of Melissa leaf having excellent angiogenesis and MMPinhibitory activities, and a composition comprising the same.

In one aspect, the present application is directed to an ethyl acetatefraction of Melissa leaf, having angiogenesis and MMP inhibitoryactivities. The Melissa leaf may be extracted with 50˜100% C₁˜C₆alcohol, and concentrated, and then the concentrated alcohol extract maybe suspended in water, and fractionated with ethyl acetate, and dried toobtain the ethyl acetate fraction of Melissa leaf. The C₁˜C₆ alcohol maybe 70-80% C₁˜C₆ alcohol. The C₁˜C₆ alcohol may be ethanol or methanol.

In another aspect, the invention is directed to a composition for theprevention or treatment of obesity, including the ethyl acetate fractionof Melissa leaf described above. The composition may be used for theprevention or treatment of diabetic retinopathy, macular degeneration(including age-related macular degeneration), pterygium, retinaldegeneration, corneal graft neovascularization, neovascular glaucoma,neovascular corneal disease, retrolental fibroplasia, trachoma, cornealulcer, proliferative vitreoretinopathy, immature retinopathy, ocularinflammation, keratoconus, sjogren's syndrome, myopia, ocular tumor orgraft rejection after keratoplasty. Prevention or treatment of cancergrowth and metastasis, may also occur, as with prevention or treatmentof haemangioma, angiofibroma, vascular malformation, arteriosclerosis,vascular adhesion, scleroderma, or restenosis. Prevention or treatmentof arthritis, rheumatoid arthritis, osteoarthritis, septic arthritis,MMP-mediated osteopenia, or degenerative cartilage loss followingtraumatic joint injury may also be carried out by the use of the ethylacetate extract of Melissa described above.

In another aspect, the invention is directed to a composition for theprevention or treatment of inflammatory diseases of the central nervoussystem or inflammatory bowel disease using the ethyl acetate fraction ofMelissa leaf described above. Prevention or treatment of psoriasis,telangiectasia, pyogenic granuloma, seborrheic dermatitis, or acne,using the ethyl acetate fraction of Melissa leaf described above isencompassed in the present invention. Prevention or treatment ofAlzheimer's disease is also included. Prevention or treatment ofabnormal wound concrescence, proteinuria, abdominal aortic aneurysm,demyelinated nerve tissue, liver fibrosis, nephroglomerular diseases,premature rupture of the fetal membrane, or periodontal diseases is alsoincluded. The inventive composition may be formulated in a dosage formof granule, powder, tablet, coated tablet, capsule, pill, syrup, drop,liquid, solution, suspension, emulsion, or injectable solutions.

These and other objects of the invention will be more fully understoodfrom the following description of the invention, the referenced drawingsattached hereto and the claims appended hereto.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will become more fully understood from thedetailed description given herein below, and the accompanying drawingswhich are given by way of illustration only, and thus are not limitativeof the present invention, and wherein;

FIG. 1A and FIG. 1B are graphs showing the inhibitory effect ofALS-L1023 on MMP activity;

FIG. 2 is a photograph showing the inhibitory effect of ALS-L1023 onHUVEC tube formation;

FIG. 3 is a graph that shows the result of mouse Matrigel assay foranalyzing the inhibitory effect of ALS-L1023 on angiogenesis;

FIG. 4 is microscopical photographs of liver tissue showing thereduction in hepatic steatosis and inflammatory cell infiltration uponfeeding high fat diet-induced obese rats with a high dose of ALS-L1023compared with the vehicle control group;

FIG. 5 is a graph showing the reduction of adipocyte area inretroperitoneal adipose tissue upon feeding high fat diet-induced obeserats with a medium and high dose of ALS-L1023 compared with the vehiclecontrol group;

FIG. 6 is a photograph showing the inhibitory effect of ALS-L1023 onprogression of exudative age-related macular degeneration by oraladministration of ALS-L1023;

FIG. 7 is a graph showing the changes in rat body weight according tothe toxicity test by a single oral dose at 2000 mg/kg of ALS-L1023;

FIG. 8 is a graph showing the changes in rat body temperature accordingto the toxicity test by a single oral dose at 2000 mg/kg of ALS-L1023;and

FIG. 9 is a graph showing the relative organ weight values, shown as apercentage of the total body weight according to the toxicity test by asingle oral dose at 2000 mg/kg of ALS-L1023.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In the present application, “a” and “an” are used to refer to bothsingle and a plurality of objects.

The present invention provides an ethyl acetate fraction of Melissa leafhaving excellent angiogenesis and matrix metalloproteinases (MMPs)inhibitory activities. In one specific embodiment of the invention, analcohol extract, preferably 75% ethanol, of Melissa yielded advantageousanti-angiogenesis effects compared with the water extract of Melissa.Moreover, further fractionating the alcohol extract of Melissa withethyl acetate resulted in an extract having even greateranti-angiogenesis and MMP inhibiting effects compared with Melissapreparations of alcohol extraction alone. Preferably, the extract ofMelissa obtained by first alcohol extraction and then ethyl acetatefractionation may be used to prevent or treat disorders or diseasescaused by angiogenesis. Thus, in one aspect, the present invention isalso directed to providing a composition comprising the ethyl acetatefraction of Melissa leaf for the treatment or prevention ofangiogenesis-related diseases and MMP-mediated diseases.

Hereinafter, the present invention is described in detail.

The ethyl acetate fraction of Melissa leaf according to the presentinvention is characterized in that Melissa leaf is extracted with50˜100% C₁˜C₆ alcohol, and concentrated, and then the concentratedalcohol extract is suspended in water, and fractionated with ethylacetate, and dried to obtain the ethyl acetate fraction of Melissa leaf.

In the method of producing an ethyl acetate fraction of Melissa leafaccording to the present invention, dried or non-dried or the mixture ofMelissa leaf can be used. For the effective extraction, Melissa leaf canbe cut into small pieces or pulverized.

An alcohol extract of Melissa leaf can be extracted by conventionalmethods. The used extraction solvent may be 50˜100% alcohol, preferably70˜80% alcohol of 5˜10 volume to Melissa leaf. The alcohol may be C₁˜C₆alcohol, preferably methanol, ethanol or a mixture thereof.

In the examples of the present invention, 75% ethanol extract of Melissaleaf is suspended in water, and fractionated with ethyl acetate, anddried to obtain the ethyl acetate fraction of Melissa leaf (calledALS-L1023).

Water-soluble materials and water-insoluble materials can be extractedeffectively by the use of 50˜100% alcohol as extraction solvent. Andthis method is effective to obtain water-insoluble material which issoluble in ethyl acetate.

Ethyl acetate is selected as a second extraction solvent consideringproduct yield, toxicity of residues and the relative content ofreference substances.

For the mass production of the fraction from Melissa leaf with excellentanti-angiogenic activity, 50˜100% alcohol extract of Melissa leaf issuspended in water, and fractionated with ethyl acetate, and dried toobtain the ethyl acetate fraction of Melissa leaf (called ALS-L1023 inthe following examples).

Also, Melissa leaf may be extracted with 50˜100% C₁˜C₆ alcohol, dried,and the alcohol extract suspended in water, and then fractionated withethyl acetate, dried, and the ethyl acetate fraction resuspended inwater, and dried to obtain the ethyl acetate fraction of Melissa leaf.

The ethyl acetate fraction of Melissa leaf obtained was used in animaltest and preclinical test.

The inventors discovered that an ethyl acetate fraction of Melissa leafaccording to this invention is excellent in inhibiting angiogenesis andMMP activities comparing with other fractions obtained by other solventpartition. The ethyl acetate fraction of Melissa leaf shows the mosteffective and excellent activities in angiogenesis inhibition throughMMP inhibition assay, HUVEC tube formation assay, and mouse Matrigelimplant assay. The inventors also found that this fraction inhibitedobesity by reducing adipose tissue, adipose cell size and inducing geneexpression related to fatty acid oxidation.

It is therefore clear that an ethyl acetate fraction of Melissa leafaccording to the present invention can be used as an anti-angiogenicagent and MMP-inhibitory agent for the treatment or prevention ofangiogenesis-related diseases and MMP-mediated diseases.

Therefore the present invention provides a composition comprising anethyl acetate fraction of Melissa leaf.

The angiogenesis-related diseases and MMP-mediated diseases that can betreated or prevented by the composition of the present inventioninclude, but are not limited to, cancer growth and metastasis; angioma,angiofibroma, vascular deformity, and cardiovascular diseases such asatherosclerosis, angiostenosis, edemic sclerosis and stenosis;opthalmological diseases such as diabetic retinopathy, maculardegeneration (including age-related macular degeneration), pterygium,retinal degeneration, angiogenesis in corneal implantation, angiogenicglaucoma, angiogenic corneal disease such as corneal synechia and irissynechia, retrolental fibroplasias, granular conjunctivitis, cornealulcer, proliferate vitreous body retinopathy, immature retinopathy, eyeinflammation in eyes, conical cornea, Sjogren's syndrome, myopia, eyestumors and rejection of cornea implantation; obesity; chronicinflammatory diseases such as arthritis, rheumatoid arthritis,osteoarthritis, septic arthritis, inflammation diseases such asinflammation of the central nervous system and inflammatory boweldisease; MMP-mediated osteopathy and regressive cartilage loss;dermatological disease such as psoriasis, telangiectasis, pyogenicgranuloma, seborrheic dermatitis and acne; Alzheimer's disease; cornealsynechia, proteinuria, abdominal aortic aneurysm, demyelinated nervetissue, liver fibrosis, nephroglomerular disease, premature rupture ofthe fetal membrane, periodontal diseases such as gingivitis andperiodontitis.

The composition of the present invention can be used in combination withknown anti-angiogenic agents or known MMP inhibitors.

In the present invention, the composition comprising the ethyl acetatefraction of Melissa leaf which is used as an agent for the treatment orprevention of angiogenesis-related diseases and MMP-mediated diseases,can be pharmaceutical composition or food composition.

The pharmaceutical composition of the present invention can alsocomprise pharmaceutically and physiologically acceptable additives suchas diluent, dispersing agent, surfactant, solvent, disintegrating agent,sweetener, binder, coating agent, blowing agents, lubricants, glidantsor flavoring agent.

The pharmaceutical composition comprising the ethyl acetate fraction ofMelissa leaf of the present invention as an active ingredient can beformulated in combination with pharmaceutically acceptable excipients,carriers or diluents.

The pharmaceutical composition of the present invention can beformulated in any form such as granule, powder, tablet, coated tablet,capsule, pill, syrup, drop, liquid, solution, suspension, emulsion, orinjectable solutions.

For example, in the composition of tablet or capsule type, the activeingredients can be bind with pharmaceutically acceptable inactive andnon-toxic carriers. And adequate binders, lubricants, disintegrants andcolor formers can be included in case of need. Examples of suitablebinders include, but are not limited to, starch, gelatin, dextrin,maltodextrin, natural sugar such as glucose or β-lactose, cornsweetener, acacia, natural and synthetic gum such as tragacanth andsodium oleate, sodium stearate, magnesium stearate, sodium benzoate,sodium acetate, sodium chloride. Disintegrants include, but are notlimited to, starch, methylcellulose, agar, bentonite, xanthan gum etc.

In the composition of liquid solution type, pharmaceutically acceptablecarriers are saline, sterile water, Ringer's solution, buffered saline,albumin injection solution, dextrose solution, maltodextrin solution,glycerol, ethanol or mixed solution. And general additives likeantioxidants, buffered solution, bacteriostat, etc. can be added to thiscomposition. By using conventional method or the written text ofRemington's pharmaceutical Science (Mack Publishing co, Easton Pa.), thecomposition of the present composition can be formulated in anydesirable forms according to disease or ingredient.

Also, the food composition comprising the ethyl acetate fraction ofMelissa leaf of the present invention as an active ingredient can beused as functional foods, dietary supplements or food additives. In caseof food additives, the food composition of the present invention can beadded to meat, drinking water, chocolate, groceries, snack, pizza,instant noodle, noodles, chewing gum, ice-cream, alcoholic beverage,vitamin complex, or healthy food.

The present invention provides the use of a composition comprising anethyl acetate fraction of Melissa leaf for the treatment or preventionof angiogenesis-related diseases and MMP-mediated diseases. Thecomposition of the present invention can be use in food and medicine forthe treatment or prevention of angiogenesis-related diseases andMMP-mediated diseases.

The present invention also provides method for the treatment orprevention of angiogenesis-related diseases and MMP-mediated diseasescomprising administering therapeutically effective amount of the ethylacetate fraction of Melissa leaf to a mammal.

As used herein, “mammal” for purposes of treatment refers to any animalclassified as a mammal, preferably, the mammal is human.

As used herein, “therapeutically effective amount” means an amount thatwill induce a biological or medical response in the animal or human, towhich it is administered. An ordinarily skilled medical provider candetermine the therapeutically effective amount, as well as, theappropriate dose and frequency of administration(s) to achieve anoptimum clinical result. A therapeutically effective amount will varydepending on various factors such as kinds of diseases, severity of thepatient's symptoms, contents of ingredients, age, body weight, sex ofthe individual patient, food, administration time, administration route,the ratio of the composition, treatment period, and other coadministereddrugs. The composition of the present invention can be administered in asingle dose, or as part of an administration regime such as multipledoses, desirable dosage of ethyl acetate fraction of Melissa leaf can be3 mg/kg-250 mg/kg per day.

The composition of the present invention can be administered by variousroutes, for example, but without limitation, orally, rectally,intravenously, intraarterially, intraperitoneally, intramuscularly,intrarmucosally, subcutaneously, intradermally, transdermally,transcutaneously, intravaginally, intrarectally, nasally, ocularly,and/or intestinally.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingfigures. Such modifications are intended to fall within the scope of theappended claims. The following examples are offered by way ofillustration of the present invention, and not by way of limitation.

EXAMPLES Example 1—Production of Ethyl Acetate Fraction of Melissa Leaf

To produce Melissa leaf extract with potent anti-angiogenic activity,the Melissa leaf is extracted with distilled water, 50% aqueous ethanol,75% aqueous ethanol, 100% ethanol and methanol, respectively. The crudeextract was centrifuged, filtered, concentrated and then lyophilized toobtain an extract powder. The powder is stored at 4° C. until use. Theanti-angiogenic effect of the extracts was tested by HUVEC tubeformation assay at the concentration of 50 μg/M

. As shown in Table 1, all the extract including water, 50%, 75%, 100%ethanol, and 100% methanol extracts showed inhibition effect in HUVECtube formation assay. Especially, the 75% ethanol extract had relativelygood inhibitiory activity comparing with other extracts, therefore 75%ethanol is selected as a primary extraction solvent.

TABLE 1 The effect of Melissa extracts on HUVEC tube formation SamplesHUVEC tube formation inhibition Negative control − Water extract + 50%Ethanol extract (+) 75% Ethanol extract +(+) 100% Ethanol extract + 100%Methanol extract +(+) −: tubes formed(no inhibition), +/−: tubes almostresemble control(rare inhibition), +: tubes are slightlydisconnected(light inhibition), ++: tubes are heavilydisconnected(significant inhibition), +++: no tubes formed.

To find the most potent anti-angiogenic activity from Melissa leaffractions, the 75% ethanol extract of Melissa leaf is sequentiallypartitioned with hexane, ethyl acetate, and butanol. Each fraction iscentrifuged, filtered, concentrated and then lyophilized to obtain afraction powder. The powder is stored at 4° C. until use. Theanti-angiogenic effect was tested by HUVEC tube formation assay at theconcentration of 50 μg/m

, respectively. As shown in Table 2, the ethyl acetate fraction has themost potent in anti-angiogenic activity.

TABLE 2 The effect of fractions on HUVEC tube formation Samples HUVECtube formation inhibition Negative control − 75% Ethanol extract +(+)Hexane fraction +/− Ethyl acetate fraction ++(+) Butanol fraction + −:tubes formed(no inhibition), +/−: tubes almost resemble control(rareinhibition), +: tubes are slightly disconnected(light inhibition), ++:tubes are heavily disconnected(significant inhibition), +++: no tubesformed.

Based on the results of these assay, mass production of Melissa leaffraction with excellent anti-angiogenic activity was performed asfollows; 75% ethanol extract of Melissa leaf is suspended in water, andfractionated with ethyl acetate, and dried to obtain the ethyl acetatefraction of Melissa leaf (called ALS-L1023 in the following examples).This ALS-L1023 was used for animal test and preclinical test.

Detailed mass production procedure of ALS-L1023 is described below.

The dried 100 kg of Melissa leaf was extracted twice with 350 L of 75%aqueous ethanol at 83° C. for 4 hours. The extract was filtered with 10μm filter and concentrated to 30 L and then fractionated with 40 L ethylacetate two times. After washing this fraction with distilled water, itwas concentrated to the volume 15 L and then dried with hot wind at 45°C. Finally, the 5 kg of ALS-L1023 was obtained in a dried powder form.

Example 2—Production of Ethyl Acetate Fraction of Melissa Leaf

(1) MMP Inhibition Assay

In order to investigate MMP inhibition by ALS-L1023 produced by massproduction, MMP enzyme activity assay was performed by aspectrofluorometric method (Perkin-Elmer LS50B).

Human MMP enzymes used in the assay were prepared by recombinant proteinproduction using baculovirus system in AngioLab, Inc. Fluorometricsubstrate (Bachem Cat. No. M-2105) was used as the substrate for MMP-2and MMP-9. ALS-L1023 was added with various concentrations to thereaction buffer containing MMP enzyme and substrate, and fluorescenceintensity was measured. The IC₅₀ of ALS-L1023 for MMP-2 and MMP-9 was17.7±1.0 μg/M

and 12.3±1.4 μg/M

respectively.

The IC₅₀ of 75% ethanol extract of Melissa leaf for MMP-2 and MMP-9 was33.6±1.5 μg/M

and 26.0±1.7 μg/M

respectively. Thus it is confirmed that the MMP inhibition activity ofALS-L1023 was enhanced comparing with that of 75% ethanol extract ofMelissa leaf (FIG. 1).

(2) HUVEC Tube Formation Inhibitory Activity

The HUVEC (Human Umbilical Vein Endothelial Cell) tube formation assayis an in vitro assay that is closely related to in vivo efficacy, andthe effect of ALS-L1023 on HUVEC tube formation was investigated.

To perform the tube formation assay, HUVECs were isolated from freshlyobtained umbilical cords. Cells were cultured and identified byimmunocytochemical staining with anti-Factor VIII antibody. HUVECscultured within passage 5 were grown on Matrigel (BD Bioscience,Bedford, Mass., USA) at 37° C. in the absence or presence of differentconcentrations of ALS-L1023 for 18 hours. Tube formation was observedusing microscope. As shown in FIG. 2, ALS-L1023 produced by massproduction inhibited tube formation at 50 μg/M

. Capillary network of tubes was disconnected by ALS-L1023 whilecapillary network of tubes was observed in control. Moreover, ALS-L1023inhibited HUVEC tube formation in a dose dependent manner as shown inTable 3.

TABLE 3 Inhibition of HUVEC tube formation by ALS-L1023 sample(μg/Ml)HUVEC tube formation inhibition 0(control) − 25 + 50 ++ 100 ++

(3) Cell Cytotoxicity Test

To test the cytotoxicity of ALS-L1023 on HUVEC, 5,000-10,000 as seededin 96 well plate and ALS-L1023 of different concentrations was added toeach well. The cell viability was tested with XTT tetrazolium base(sodium3′-[1-(phenylaminocarbonyl)-3,4-tetrazoium]-bis[4-methoxy-6-nitro]benzenesulfonic acid hydrate) cell proliferation kit (Roche, Germany) andviable cells were measured by ELISA plate reader. ALS-L1023 did notaffect HUVEC viability at the concentration of 50 μg/M

showing HUVEC tube formation inhibition.

(4) Mouse Matrigel Assay

Mouse Matrigel implant assay was performed to measure the inhibition ofin vivo angiogenesis by ALS-L1023 quantitatively.

A 0.4 ml of Matrigel containing 50 ng/ml of basic fibroblast growthfactor (bFGF) and 50 units/ml of heparin was implanted by subcutaneousinjection into C57BL/6 mouse. 0.5 mg of ALS-L1023 dissolved in 10%ethanol per mouse was orally administered twice a day for 4 days. 10%ethanol was orally administered to control group. At day 5, an epidermisof mouse was removed, and Matrigel was recovered, and then the amount ofhemoglobin in the Matrigel was determined by Drabkin's reagent (Sigma,USA).

As shown in FIG. 3, the hemoglobin contents in Matrigel recovered fromALS-L1023 treated group were decreased as compared with the vehiclecontrol group. The percent of angiogenesis inhibition by ALS-L1023 was33%.

FORMULATION EXAMPLES

Suitable excipients were added to ALS-L1023 to improve its stability,and then 250 mg thereof was packed in a hard capsule. The mixing ratiois described in the following Table 4.

TABLE 4 Formulation ratio of ALS-L1023 Raw material mixing ratio (%)ALS-L1023 60 Magnesium stearate 1 Colloidal silicon dioxide 1Microcrystalline cellulose 33 Sodium lauryl sulfate 5

Example 3—Anti-Obesity Effect of ALS-L1023 Fraction

With respect to high fat diet-induced obesity, the following experimentwas performed to examine the inhibitory effect of ALS-L1023 on abdominalfat. The formulated ALS-L1023 as an active ingredient was mixed with a45 kcal % high fat feed (manufactured by Research Diets Inc., USA) in acontent of 0 (control group), 0.1% (low), 0.25% (medium), and 0.5%(high) (formulated form: 0, 0.17, 0.42, 0.83%). For the preparation ofthe control group containing no active ingredient ALS-L1023, excipientscontained in the formulated ALS-L1023 were mixed with the high fat feedin a content of 0.33% as a high dose. After a one-week acclimationperiod, 7-week-old male SD rats were divided into groups of 7 mice pergroup, and fed for 12 weeks. Then, their body weight and weight ofabdominal fat were measured, and biochemical blood tests andhistopathological tests of liver and adipose tissue were performed.

(1) Body Weight

Body weight was measured on the day of acquisition, initiation day ofadministration, once every week after initiation of the administration,and the day of autopsy. The results are summarized in Table 5, in whichthe mean body weight of the vehicle control group constantly increasedfrom 204.73±5.57 g (before administration) to 633.63±43.08 g (for 12weeks after administration), and the mean body weight of ALS-L1023 group(low, medium, and high dose groups) constantly increased from210.10±5.19 g, 204.97±6.61 g, and 208.01±7.65 g (before administration)to 623.72±58.02 g, 588.69±33.76 g, and 584.83±33.44 g (for 12 weeksafter administration). There was no statistical significance between thevehicle control and ALS-L1023 groups. However, the mean body weight ofALS-L1023 group decreased in a dose-dependent manner.

TABLE 5 Changes in body weight by administration of ALS-L1023 0 0.1 0.250.5 ALS-L1023 (control (low dose (medium dose (high dose content (%)group) group) group) group) Body weight 204.73 ± 5.57  210.10 ± 5.19 204.97 ± 6.61  208.01 ± 7.65  before administration (g) Body weight633.63 ± 43.08 623.72 ± 58.02 588.69 ± 33.76 584.83 ± 33.44 afteradministration (g)

(2) Feed Intake

Daily feed intake was determined as follow. The feeder was filled withpowdery feed, and then its weight was measured. At 24 hrs after feedingthe animals with the feeder, the weights were measured, and a differencein the measured weights was determined as a daily feed intake. Themeasurement was performed on the initiation day of the administration,and once every week after initiation of the administration.

Mean daily feed intake of the vehicle control group was within a rangeof 18.46 to 22.73 g for 12 weeks, and mean daily feed intakes of low,medium, and high dose groups were within a range of 17.14 to 21.84 g,17.29 to 22.03 g and 16.99 to 20.84 g, respectively. There was nosignificant difference, as compared with the vehicle control group.

(3) Organ and Adipose Tissue Weights

Before autopsy and blood collection, the animals were fasted for aperiod of 18 hrs or more, and anesthetized with ether, followed by bloodcollection and phlebotomy. The mesenteric adipose tissues, epididymaladipose tissues, and retroperitoneal adipose tissues were excised fromthe abdominal cavity, and weighed. The liver, heart, kidney, spleen, andpancreas were weighed.

As a result, there was no significant difference in the weights ofheart, liver, pancreas, kidney (left, right) and spleen between thevehicle control and experimental groups. With respect to the mesentericadipose tissue weight, the low, medium and high dose groups were15.90±4.36 g, 12.64±1.69 g, and 11.23±3.32 g, respectively. That is, theexperimental groups were decreased in a dose-dependent manner, comparedwith the vehicle control group of 15.16±4.41 g. With respect toepididymal adipose tissue weight, the low, medium and high dose groupswere 17.74±3.37 g, 16.29±2.62 g, and 15.67±3.83 g, respectively. Thatis, the experimental groups were decreased in a dose-dependent manner,compared with the vehicle control group of 20.80±4.41 g, and astatistically significant difference (p<0.05) was observed in the highdose group. With respect to retroperitoneal adipose tissue weight, thelow, medium and high dose groups were 27.20±5.76 g, 21.36±4.03 g, and23.94±6.24 g, respectively. That is, the experimental groups weredecreased in a dose-dependent manner, compared with the vehicle controlgroup of 29.73±3.23 g, and a statistically significant difference(p<0.05) was observed in the medium dose group. With respect to a totalweight of the abdominal adipose tissue including the mesenteric adiposetissue, the epididymal adipose tissue and the retroperitoneal adiposetissue, the low, medium and high dose groups were 60.84±11.66 g,50.29±7.31 g, and 50.84±11.84 g, respectively. That is, the experimentalgroups were decreased, compared with the vehicle control group of65.69±10.13 g, and a statistically significant difference (p<0.05) wasobserved in the medium and high dose groups (see Table 6).

TABLE 6 Changes in organ and adipose tissue weights by administration ofALS-L1023 0 0.1 0.25 0.5 ALS-L1023 (control (low dose (medium dose (highdose content (%) group) group) group) group) mesenteric 15.16 ± 4.4115.90 ± 4.36 12.64 ± 1.69  11.23 ± 3.32 adipose tissue weight (g)epididymal 20.80 ± 4.41 17.74 ± 3.37 16.29 ± 2.62   15.67 ± 3.83**adipose tissue weight (g) retroperitoneal 29.73 ± 3.23 27.20 ± 5.7621.36 ± 4.03** 23.94 ± 6.24 adipose tissue weight (g) total weight of 65.69 ± 10.13  60.84 ± 11.66 50.29 ± 7.31**   50.84 ± 11.84** abdominaladipose tissue (g) **p < 0.05 compared with the control group

(4) Biochemical Blood Tests

In the biochemical blood tests, there was no significant difference inliver indices, such as serum levels of AST (Aspartate aminotransferase),ALT (Alanine aminotransferase), and ALP (Alkaline phosphatase) betweenthe control and experimental groups. With respect to total bloodcholesterol level, the low, medium and high dose groups were 88.07±14.35mg/dL, 86.96±17.68 mg/dL, and 79.90±10.45 mg/dL, respectively. That is,the experimental groups were decreased in a dose-dependent manner,compared with the vehicle control group of 102.09±21.82 mg/dL. Withrespect to triglyceride level, the low, medium and high dose groups were50.93±15.28 mg/dL, 46.63±11.77 mg/dL, and 45.50±22.45 mg/dL,respectively. That is, the experimental groups were also decreased in adose-dependent manner, compared with the vehicle control group of58.13±25.04 mg/dL. With respect to LDL level, the low, medium and highdose groups were 9.56±2.67 mg/dL, 9.11±2.92 mg/dL, and 8.56±2.26 mg/dL,respectively. That is, the experimental groups were also decreased in adose-dependent manner, compared with the vehicle control group of12.31±3.70 mg/dL. However, there was no significant difference in thelevels of glucose, total protein, and albumin between the control andexperimental groups. Thus, with regard to ALS-L1023 administration, theserum levels in total cholesterol, triglyceride, and LDL of theexperimental groups were decreased in a dose-dependent manner, comparedwith the vehicle control group (see Table 7).

TABLE 7 Changes in blood biochemical indices by administration ofALS-L1023 0 0.1 0.25 0.5 ALS-L1023 (control (low dose (medium dose (highdose content (%) group) group) group) group) total cholesterol 102.09 ±21.82 88.07 ± 14.35 86.96 ± 17.68 79.90 ± 10.45 (mg/dL) triglyceride 58.13 ± 25.04 50.93 ± 15.28 46.63 ± 11.77 45.50 ± 22.45 (mg/dL) LDL(mg/dL) 12.31 ± 3.70 9.56 ± 2.67 9.11 ± 2.92 8.56 ± 2.26

(5) Histopathological Test of Liver

The livers were weighed, and then fixed in 10% neutral buffered formalinsolution, followed by routine histological processing. Tissue slidesamples were prepared, and then stained with H&E, followed byobservation under a microscope for hepatic steatosis. As a result, inthe vehicle control group, excessive accumulation of fat globules wasobserved in hepatic lobule of hepatocyte, which is the finding ofhepatic steatosis. The microvesicular or macrovesicular fatty changeswere mainly observed around the hepatic portal vein. Inflammatory cellinfiltration that occurs at an advanced stage of hepatic steatosismainly occurred between or around ballooned hepatocytes where hepaticsteatosis occurred. At a moderate grade of hepatic steatosis,inflammatory cell infiltration occurred around central veins. Otherabnormal pathological findings were not observed.

In the low dose group, hepatic steatosis was also observed, which wassimilar to that observed in the vehicle control group. In the mediumdose group, the slightly decreased hepatic steatosis and inflammatorycell infiltration were observed, as compared with the vehicle controland low dose groups.

In the high dose group, the inhibition of hepatic steatosis or decreasedinflammatory cell infiltration was observed, which was similar to thatobserved in the medium dose group (see FIG. 4). This result suggeststhat ALS-L1023 exerted the inhibitory effects thereon.

(6) Adipocyte Area in Adipose Tissue

The excised retroperitoneal adipose tissues were weighed, and thenparaffin-embedded tissue samples were prepared by routine histologicprocessing, followed by H&E staining. 5 sampling sites were randomlychosen, and mean value of 10 adipocyte areas adjacent to each samplingsite were determined as an adipocyte area of each subject using stereoinvestigator software (MicroBrigthField, VT, USA) connected to anoptical microscope. As shown in FIG. 5, the reduction in the adipocytearea was observed in the medium and high dose groups, as compared withthe vehicle control group. A statistically significant difference(p<0.05) was observed in the high dose group.

Taken together, when high fat diet-induced obese rats were fed withALS-L1023 for 12 weeks, ALS-L1023 exerted inhibitory effects of obesity,including the reduction in the abdominal adipose tissue weight and inthe serum levels of total cholesterol and triglyceride, the alleviationof hepatic steatosis, and the reduction in adipocyte size.

Example 4—Effect of ALS-L1023 on Neovascular Ocular Diseases

In order to evaluate the efficacy of ALS-L1023 on retinalneovascularization, Oxygen-induced retinopathy mice in ROP (retinopathyof prematurity) animal model were intraperitoneally injected withdiluted ALS-L1023 in DMSO at a dose of 25 mg/kg on day 13 after birthfor a period of 5 days.

As a result, retinal neovascularization and many abnormal blood vesselswere observed in the ROP control group, whereas normal retinas showuniform and compact patterns of blood vessels. Notably, reducedneovascularization in the central retina and peripheral retina and thelower number of abnormal blood vessels were observed in the ALS-L1023group, as compared with the ROP control group. Thus, ALS-L1023 isexpected to be effective in preventing and treating the ocularneovascularization.

Example 5—Effect of ALS-L1023 on Macular Degeneration

The formulated ALS-L1023 capsule was orally given to a male patient whowas diagnosed with the exudative age-related macular degeneration threetimes daily with 2 capsules on each occasion for 12 weeks. Then, themacular area of the patient was photographed, and the size of maculardegeneration area was compared with its photograph taken beforeadministration. As shown in FIG. 6, no progression was observed in themacular degeneration area. Thus, ALS-L1023 is expected to be effectivein preventing and treating the age-related macular degeneration.

Experimental Example 6—Safety Test of ALS-L1023

Melissa leaf, from which the test substance ALS-L1023 was isolated, hasbeen used as a food or medicine over a long period of time, andclassified by US FDA as GRAS (generally regarded as safe). Thus, thetest substance ALS-L1023 was considered as non-toxic, and the presentexperiment was performed as an acute toxicity test (limit test) by asingle oral dose at 2000 mg/kg of ALS-L1023 in accordance with the OECDguidelines.

Ten male and ten female rats were randomly divided into the control andexperimental groups, each consisting of five animals, respectively. Thecontrol groups were administered with 1 ml/150 g (body weight) of cornoil, and the experimental groups were administered with 2000 mg/kg ofALS-L1023 in corn oil. Since the test substance ALS-L1023 was notdissolved in water, it was suspended in corn oil to be used for thetest. The test substance was prepared on the day of administration, andsuspended by vortexing prior to administration.

First, one rat was orally administered, followed by observation for 24hrs. Next, two rats were orally administered, followed by observationfor 72 hrs. Then, the rest 17 rats were orally administered, andobserved for toxicity findings for 30 min. Thereafter, the observationswere performed every 30 min for 4 hrs. Subsequently, the observationswere performed twice a day for 14 days, and body weight and temperaturewere recorded every day.

On the last day of the experiment, the rats were euthanized using CO₂and autopsied. Abnormal tissues examined by the pathologist's view wereexcised, and subjected to histological examination.

As a result, in the case of a single oral dose at 2000 mg/kg ofALS-L1023 into each of the five male and female rats, no significanttoxicity findings were observed over the course of the 14 dayobservation period.

In both control and experimental groups, distinct clinical abnormalitieswere not observed. However, one female rat became slightly excited at 60min after administration, but maintained stable at 90 min afteradministration.

In addition, ALS-L1023 administration did not affect the body weight andtemperature in both control and experimental groups (see FIGS. 7 and 8).

Relative weights (%) of thymus, heart, spleen, kidney, liver, and brainwith respect to the total body weight are shown in FIG. 9. A slight, butstatistically significant increase was observed in the liver weight ofthe female rat in the control group, as compared with that of the femalerat administered with ALS-L1023, but not in that of male rats. Abnormalautopsy findings and other peculiar findings were not found.

Moreover, although some abnormal findings were detected by thepathologist's view, histological examination thereof showed no evidenceof abnormality.

Consequently, a single oral dose at 2000 mg/kg of ALS-L1023 did notinduce any toxic symptoms over the course of the observation period.

An ethyl acetate fraction of Melissa leaf of the present invention hasstrong and excellent anti-angiogenic and MMP inhibitory activities.Therefore, the composition comprising the ethyl acetate fraction ofMelissa leaf of the present invention can be used as an agent for thetreatment or prevention of angiogenesis-related diseases andMMP-mediated diseases.

All of the references cited herein are incorporated by reference intheir entirety.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention specifically described herein. Suchequivalents are intended to be encompassed in the scope of the claims.

1.-17. (canceled)
 18. A method of reducing neovascular ocular diseasesor treating age-related macular degeneration (AMD) in a subject,comprising administering to a subject in need thereof Melissa leafextract, which has been extracted with 75% ethanol, and concentrated,and then the concentrated alcohol extract is suspended in water, andfractionated with ethyl acetate, and dried to obtain the ethyl acetatefraction of Melissa leaf.
 19. A method of reducing neovascular oculardiseases or treating age-related macular degeneration (AMD) in asubject, comprising (i) extracting Melissa leaf with 75% ethanol; (ii)concentrating the alcohol extract; (iii) suspending the concentratedalcohol extract in water; (iv) fractionating the alcohol extract inwater with ethyl acetate; (v) drying the ethyl acetate fraction toobtain the ethyl acetate fraction of Melissa leaf; and administering toa subject in need thereof the obtained ethyl acetate fraction of Melissaleaf.